FIGURE 5.

Structure probing of the BM2 termination–reinitiation signal. RNA derived by transcription of p2luc-BM2-T3/Bam HI with T3 RNA polymerase was 5′-end labeled with [γ-³³P]-ATP and subjected to limited RNase or chemical cleavage using structure-specific probes. Sites of cleavage were identified by comparison with a ladder of bands created by limited alkaline hydrolysis of the RNA (OH−) and the position of known RNase U2 and T1 cuts, determined empirically. Products were analyzed on a 10% acrylamide/7 M urea gel containing formamide. Data also were collected from 6% and 15% gels (gels not shown). Enzymatic structure probing was with RNases CL3, T1, U2, and CV1. Uniquely cleaved nucleotides were identified by their absence in untreated control lanes (0). The number of units of enzyme added to each reaction is indicated. Chemical structure probing was with imidazole (4 h, I) or lead acetate (Pb²⁺; millimolar concentration in reaction). The water lane (W) represents RNA that was dissolved in water, incubated for 4 h and processed in parallel to the imidazole-treated sample. The sequence of the probed RNA and the inferred secondary structure is shown in Figure 6.